Identification of three Asian otter species (Aonyx cinereus, Lutra sumatrana, and Lutrogale perspicillata) using a novel noninvasive PCR‐RFLP analysis

Abstract Four species of otters occur in tropical Asia, and all face multiple threats to their survival. Studies of distribution and population trends of these otter species in Asia, where they occur sympatrically, are complicated by their elusive nature and difficulties with reliable identification of species in field surveys. In Malaysia, only three species, the smooth‐coated otter, Asian small‐clawed otter, and hairy‐nosed otter have been reliably reported as residents. We designed a replicable and cost‐efficient PCR‐RFLP protocol to identify these three species. Using published reference sequences of mitochondrial regions, we designed and tested three PCR‐RFLP protocols on DNA extracted from reference samples and 33 spraints of wild otters collected along the North Central Selangor Coast of Malaysia. We amplified and sequenced two fragments (450 and 200 bp) of the mt D‐loop region and a 300‐bp fragment of the mt ND4 gene using primer sets TanaD, TanaD‐Mod, and OTR‐ND4, respectively. Amplification products were digested with restriction enzymes to generate species‐specific RFLP profiles. We analyzed the costs of all three protocols and compared these with the costs of sequencing for species identification. Amplification success was highest for the smallest PCR product, with the TanaD‐Mod primer amplifying DNA from all 33 spraints. TanaD and OTR‐ND4 primers amplified DNA from 60.6% and 63.6% spraints, respectively. PCR products of TanaD‐Mod provided the expected species‐specific RFLP profile for 32 (97%) of the spraints. PCR products of OTR‐ND4 provided the expected RFLP profile for all 21 samples that amplified, but TanaD produced spurious bands and inconsistent RFLP profiles. The OTR‐ND4 primer–enzyme protocol was the least expensive (437 USD) for processing 100 samples, followed by TanaD‐Mod (455 USD). We suggest the use of both OTR‐ND4 and TanaD‐Mod protocols that show potential for highly efficient and reliable species identification from noninvasive genetic sampling of three Asian otter species. We expect our novel noninvasive PCR‐RFLP analysis methods to facilitate population monitoring, ecological and behavioral studies on otters in tropical and subtropical Asia.

Among these four species, the smooth-coated otter and the Asian small-clawed otter are listed as Vulnerable, the hairy-nosed otter as Endangered, and the Eurasian otter as Near-Threatened in the IUCN's Red List assessment (Khoo et al., 2021;Roos et al., 2021;Sasaki et al., 2021;Wright et al., 2021). There is a dearth of information about the distribution and conservation status of these four species of otters in South-East Asia (Duplaix & Savage, 2018), where populations are threatened by rapid economic development, habitat loss, illegal wildlife trade, pollution and degradation of aquatic habitat, human-otter conflict, and climate change (Cianfrani et al., 2018;Duckworth & Hill, 2008;Foster-Turley, 1992). Increased pollution of aquatic habitats has impacted otter food resources and expanding linear infrastructure such as road networks is emerging as one of the major causes of otter mortality (Peterson & Schulte, 2016).
Increasing threats to otter populations throughout the world have called for increased and focused efforts to monitor their status and distributions (Duplaix & Savage, 2018). However, due to their elusive habits and rarity in distribution, monitoring otters is not an easy task. A plethora of noninvasive survey techniques, which possess the advantages of being relatively inexpensive and replicable, exist to monitor small carnivores (Long et al., 2012). Sign surveys using footprints and spraints have been traditionally used for monitoring presence, population trends, and habitat use, specifically for the Eurasian otter and North American River otter (Lontra canadensis). Otter spraints are typically deposited at latrine sites located along the banks of rivers and wetlands. Fresh otter spraints are easily detected by their strong fishy odor. In the last two decades, advancements in camera trapping and noninvasive DNA analysis techniques have opened new avenues for otter monitoring. Camera traps at latrine sites have provided valuable data on species, group size, and social behavior (Ben-David et al., 1998;Green et al., 2015;Stevens & Serfass, 2008). Noninvasive molecular genetic sampling using spraints has been successfully employed in ecological and demographic studies of Eurasian and North American otters (Hájková et al., 2009;Jansman et al., 2001;Klütsch & Thomas, 2018;Sittenthaler et al., 2020). These methods increase the scope of population monitoring by providing data on otter presence and habitat use, including additional information such as individual identification by DNA analysis, which can be used to estimate population size and density.
Accurate species identification is vital for population monitoring.
However, where the ranges of multiple species of otters overlap, as in South-East Asia, surveys using sign or camera traps are limited by uncertainty and difficulties in definitively assigning spraints, footprints, or photographs to species. Kruuk et al. (1993) and Kistner et al. (2022) provide detailed descriptions to distinguish among tracks, spraints, and spraint sites of three otter species (Lu. lutra, A. cinereus, and L. perspicillata), but these signs, specifically tracks, persist for a short period in tropical weather conditions, and their reliable interpretation depends largely on the consistency of the substrate. A majority of South Asian and South-East Asian countries have more than two otter species. Cambodia, Indonesia (Sumatra), Thailand, Myanmar, and Vietnam report four species. Thus, efficient and cost-effective methods to overcome the challenge of species identification are crucial for monitoring otter populations in these regions.
Although all four species of otters have been reported from Malaysia, only three species, the smooth-coated otter, Asian smallclawed otter, and hairy-nosed otter, have consistently confirmed distribution records. Smooth-coated otters and Asian small-clawed otters are the most common and occur through much of tropical Asia including Peninsular Malaysia (Abdul-Patah et al., 2020;Wright et al., 2021) and Malaysian Borneo (Forest Department Sarawak, 2020; Khoo et al., 2021). The hairy-nosed otter is the only species of otter endemic to South-East Asia, but is rare with sporadic records from its known range, including Malaysia (Sasaki et al., 2021). A recent study (Abdul-Patah et al., 2020) provided the most comprehensive information on the spatial distribution, associated habitats, and genetic diversity (mitochondrial) of the three

Conservation genetics
F I G U R E 1 A group of the Asian small-clawed otter (Aonyx cinereus) in their tidal habitat of the Sundarban mangrove forest in Bangladesh. species of otters in Peninsular Malaysia. This study confirmed otter species in 138 locations of Peninsular Malaysia, out of which hairy-nosed otters were only confirmed from seven sparsely distributed locations. Roadkills of the hairy-nosed otter have been reported near peat swamp forests in Perak, Pahang (Sebastian, 1995), and more recently in Northern Selangor, Malaysia (Tan, 2015;Woo, 2021). The presence of the Eurasian otter in Malaysia is highly uncertain and limited to just two records from the northern part of the peninsula (Miller, 1900;Mohd-Azlan & Sharma, 2006) and one from Borneo (Phillipps & Phillipps, 2016 Species identification by DNA analysis of scat samples has relied mainly on the amplification of mitochondrial DNA sequences, such as the control and 16s rRNA regions (e.g., Mills et al., 2000) cytochrome b gene (e.g., Hsieh et al., 2001;Madisha et al., 2015) and mitochondrial D-loop region (Bozarth et al., 2010). DNA from noninvasive sources such as hair and feces usually contain only trace amounts of low-quality fragmented DNA, which degrades further with time, resulting in amplification failure in old decayed samples (Shih et al., 2017;Waits & Paetkau, 2005). Abdul-Patah et al. (2014 successfully identified otter species from spraints by sequencing a fragment of ~450-bp DNA, amplified from the mitochondrial D-loop region. They were successful in amplifying DNA from ~50% of the spraint samples (A.-P. Pazil, personal communication) to ascertain species. The amplification of short (<300 bp) fragments of mitochondrial DNA is reported to produce higher amplification success rates (Madisha et al., 2015;Shih et al., 2017).
PCR amplification of mitochondrial fragments has been successfully combined with restriction-fragment-length polymorphisms (PCR-RFLP) analysis to distinguish among closely related taxa (e.g., Bidlack et al., 2007;Cossíos & Angers, 2006;Hansen & Jacobsen, 1999;Mukherjee et al., 2010). This approach has the advantages of being relatively rapid and cost-effective because amplification products can be separated by size, producing distinct species-specific band patterns and eliminating the need for sequencing. Scats deposited by other small carnivores may sometimes be misidentified as otter spraints. Erroneous identification of scats of American mink (Neovison vison), European mink (Mustela lutreola), and European polecat (Mustela putorius) as those of European otter is reported (Hansen & Jacobsen, 1999), but no such information is reported by studies on Asian otter species.
Otters are apex carnivores in aquatic habitats and thus crucial for regulating aquatic food webs and ecosystem-level trophic dynamics (Ben-David et al., 1998;Kruuk, 2006). Tracking the status of otter populations is therefore fundamental to ensure the stability of aquatic ecosystems in South and South-East Asia. Conservation monitoring programs for elusive species such as otters require significant effort to achieve sufficient sample sizes for reliable estimates.
Higher amplification success rates and accurate species identification with noninvasive samples will greatly improve the quality of data needed for accurate mapping of otter species range, population distributions, estimation of occupancy, density, and population trends. Here, we develop and evaluate three PCR-RFLP (polymerase chain reaction-restriction-fragment-length polymorphism) protocols with the aim of designing an efficient and cost-effective noninvasive species identification method for three otter species found in Malaysia and other parts of South-East Asia. Further, we compare these PCR-RFLP protocols for their cost-effectiveness and their ability in successfully and reliably identifying these otter species.

| Field sampling and reference sample collection
This study is the preliminary part of a larger project to systematically Opportunistic records and surveys confirm that all three species of otter inhabit this region (Abdul-Patah et al., 2020;Woo, 2021). We collected 33 otter spraint samples from various locations in this region ( Figure 2). We obtained reference samples (blood samples on FTA cards and/or tissue samples) of two species of otters (smoothcoated otter and Asian small-clawed otter), common palm civet (Paradoxurus hermaphroditus), and short-tailed mongoose (Herpestes brachyurus) from the Department of Wildlife and National Parks, Peninsular Malaysia (PERHILITAN; Table S1). The common palm civet and short-tailed mongoose are two small carnivores that occur sympatrically with otters in the North Central Selangor Coast. Their scats are similar in size to those of otters, although chances of scat misidentification are generally minimal due to morphological dissimilarities between scats and spraints and dietary differences among these species.
We surveyed potential otter habitat to locate spraint sites and collected fresh spraints in 5-ml collection vials with absolute ethanol as preservative. These samples were stored at room temperature/−20°C before lab analysis.

| Laboratory analysis
We extracted DNA from approximately 0.25 mg of spraint samples via the alkaline lysis method using the GF-1 Soil Sample DNA Extraction Kit (Vivantis Technologies). DNA extraction procedure followed the manufacturer's instructions with a slight modification; the incubation time was increased to 5 min prior to DNA elution to maximize DNA yield. We also extracted DNA from reference samples of A. cinereus, L. perspicillata, and H. brachyurus from blood samples stored on the FTA cards and P. hermaphroditus from tissue samples using the GF-1 Tissue Blood Combi DNA Extraction Kit (Vivantis Technologies). The extracted DNA was stored at −20°C.
We amplified two fragment sizes of the mt D-loop region using two different sets of primers: (i) forward TanaD-F, 5′-ACCATCAGCACCCAAAGCTG-3′ (Masuki et al., 2008) and reverse TanaD-R, 5′-GGGCTGATTAGTCATTAGTCCATC-3′ (Masuki et al., 2008)  GenBank OL588632.1-OL588638.1; Appendix 1). We used the OligoAnalyzer™ Tool (Integrated DNA Technologies) to check for secondary structures in these primers. Thereafter, we searched for appropriate sets of restriction enzymes using an interactive web-based program VIRS (Chen et al., 2009). This tool can identify and visualize restriction sites in multiple DNA sequences and suggests appropriate restriction enzymes for those sites. Our objective was to select those enzymes that would not be affected by CpG methylation and provide discernible band patterns (ideally less than three bands with sufficient difference among their size) for easy visualization on agarose gels. We also conducted in silico PCR and restriction digestion, followed by gel visualization in the SnapGene program (GSL Biotech LLC).
DNA amplifications of the targeted fragments were conducted in a final reaction volume of 25 μl consisting of 12.5 μl ExPrime Taq Premix (2X) (GENETBIO Inc.), 7.5 μl distilled water, 1.5 μl of 10 pmol/μl forward and reverse primers, and 2 μl extracted otter fecal DNA. PCR was carried out using T100® Thermal Cycler (Bio-Rad Laboratories) with the following parameters: initial denaturation at 94°C for 10 min followed by 45 cycles of denaturation at 94°C for 30 s, annealing at 56°C and 62°C for D-loop fragments and ND4 fragment, respectively, for 30 s, and extension at 72°C for 1 min, and a final extension at 72°C for 7 min. The amplicons were electrophoresed and visualized on a 2% agarose gel to determine specific amplifications. We included extraction negatives and PCR negatives in every batch of PCR performed. First, we confirmed all reference samples, including one spraint sample of hairy-nosed otter collected from our study area that was used as a reference for all follow-up lab analysis. These samples were sequenced (Applied Biosystems™ 3730xl DNA Analyzer) for the mt Dloop and mt ND4 regions with the primers mentioned above. Forward and reverse sequences were checked, edited, and merged into contigs using ChromasPro2.0 (Technelysium Pty. Ltd.). Sequences obtained from this study were compared with reference taxonomic sequences deposited in the National Center for Biotechnology Information (NCBI) using the nucleotide Basic Local Alignment Search Tool (nBLAST) with default settings. Species-specific amplifications were confirmed if query sequences shared high similarity with any taxa from the Lutrinae subfamily. After confirming species identity, these reference samples were used as positive controls for subsequent PCR reactions of 33 spraint samples collected from various sites in the study region.
After confirming the amplification of PCR products, restriction digestions were conducted in a final reaction volume of 30 μl consisting of 10 μl PCR products, 1-2 μl restriction enzyme (Thermo Fisher Scientific), 2 μl 1X buffer (Thermo Fisher Scientific), and 16-17 μl distilled water. The digestion mixtures were incubated at 37°C (Hin1II, NdeI, and BsuRII) and 65°C (MseI and HpyCH4III) for 4 h, and digested products were separated via electrophoresis on a 2.5% agarose gel at 60 V for 2 h.

| Cost analysis
Our aim was to develop a cost-effective and accurate protocol that would be useful and financially feasible to otter researchers in Asia.
Thus, we evaluated the cost of the reagents required for processing a batch of 100 samples to determine the most cost-effective approach for species identification of three species of otters using the PCR-RFLP method with three primer sets and a combination of suitable restriction enzymes. We also compared the cost of PCR-RFLP with sequencing costs for species identification. Reagents used in this study were commercially available and were obtained from local distributors in Malaysia. We estimated and converted the cost in U.S. Dollars (US$).

| RE SULTS
Sequencing confirmed that all reference samples were from their respective species. Reference samples for three otter species, including the spraint sample of one hairy-nosed otter and the common palm civet, amplified successfully with the three primer sets (TanaD, TanaD-Mod, and OTR-ND4) used in this study and were ascertained to their respective species by an nBLAST search (Figure 3a,e,g). We Amplification rates of 33 samples differed among the three primer sets. Amplification success decreased with increasing PCR fragment size. The TanaD primer with the largest PCR product (~450 bp) amplified just 60.6% (n = 20) of DNA from spraints and produced spurious bands of various sizes for several samples. This primer set also produced inconsistent results for RFLP analysis with three different restriction enzymes MseI, NdeI, and HpyCH4III. Therefore, we discontinued the use of TanaD to analyze further samples using the PCR-RFLP method. Amplification rates were slightly better (63.6% of spraints) for the OTR-ND4 primer, which produced a band of ~300 bp. Moreover, OTR-ND4 provided the expected RFLP profile for correct species identification after RFLP analysis with BsuRI for all 21 spraint samples that successfully amplified. The TanaD-Mod primer, which yielded the smallest PCR product (~200 bp) of all three primer sets, amplified all 33 samples. Subsequent RFLP analysis with Hin1II restriction enzyme identified the species of otter in 32 (97%) of the samples.
We compared the costs of PCR-RFLP analysis for the three protocols. The OTR-ND4 primer and BsuRI enzyme pair was the least expensive, totaling 437 USD for DNA extraction, PCR amplification, and RFLP analysis of 100 samples (Table 1). The TanaD-Mod primer with Hin1II enzyme would cost 455 USD for analysis. However, if compared both for consistency and accuracy in species identification and costs, the TanaD-Mod and Hin1II pair emerges as the most cost-effective PCR-RFLP protocol (Table 2).

| DISCUSS ION
Monitoring small carnivores is a daunting task because of their elusive nature and sparse distribution. Where closely related species of small carnivores co-occur, signs such as tracks and scats are often not very useful to ascertain species. Remote cameras have improved the quality of small carnivore surveys, but have limitations where morphologically similar species such as Asian otters co-occur. There is an enormous lack of information about small carnivore distribution and ecology, with no robust and scientifically validated information on distribution ranges and population size for a number of species, owing to the lack of research studies (Glatston & Duplaix, 2020;Torres-Romero & Giordano, 2022).
Without this baseline information, assessment of the impacts and extent of threats to these species and the effectiveness of conservation measures is difficult. The primary goal of this study was to develop an accurate, easyto-implement, cost-effective DNA analysis method for the identification of three Asian otter species. We describe a cost-effective PCR-RFLP method for identifying three otter species that has very high amplification success with noninvasive samples and is half the cost of Sanger sequencing for species identification. We also detected an inverse relationship between fragment size and amplification success rates for the three primers in this study, which has also been reported in previous studies (Broquet et al., 2007;Deguilloux et al., 2002Deguilloux et al., , 2003Shih et al., 2017). Previously, the TanaD  We designed the OTR-ND4 primer for a second primer-enzyme combination targeting the mitochondrial ND4 region of the three otter species. Although this primer-enzyme pair had less amplification success than TanaD-Mod, it had moderately high accuracy and was the least expensive among all three PCR-RFLP protocols.

Lab analysis
We suggest the use of the OTR-ND4 primer in conjunction with the TanaD-Mod primer-enzyme pair as a complimentary marker where species identification needs to be reconfirmed (e.g., forensic analysis of confiscated samples).
It is prudent to mention here that hybridization between L. perspicillata and A. cinereus has been reported in the putative otter sample of L. perspicillata collected from Singapore (Moretti et al., 2017), but this study also mentions that hybridization events such as these are likely to occur when the population of one species is much smaller than the other (A. cinereus in this case). This calls for further investigation with nuclear markers to ascertain species identification, particularly if one of the co-occurring species is rare in the study region.
Cost programs for otters in Asia, we anticipate that this protocol will improve efficiency and enhance data quality.
We caution that our PCR-RFLP approach is based on a reference database consisting largely of otter species from the Malay peninsula. In other regions, different RFLP profiles may result because of regional haplotype differences. Thus, we suggest for an initial pilot study to confirm species identity via sequencing as an essential prelude to surveys using the PCR-RFLP methods described here.

| CON CLUS ION
Otters are sentinels of freshwater ecosystems. They are also resilient species that respond well to habitat conservation and population protection. In places where habitats (rivers, wetlands, and marine coastlines) are restored, and where pollution is controlled, otters have shown remarkable recolonization potential (Duplaix & Savage, 2018). Many of the world's species of otter coexist with people, including in highly urbanized areas such as Singapore (Khoo & Lee, 2020;Theng & Sivasothi, 2016). Making an active effort for otter conservation in these regions is also linked with some of the specific Sustainable Development Goals (SDGs) such as SDG 6clean water and sanitation, SDG 14-life below water, SDG 15-life on land, and SDG 13-climate action. Asian otters are in peril due to loss of habitat, trafficking for pelts and the pet trade, and from conflicts with aquaculture industries. Conservation measures for these species would require robust, replicable, and cost-effective monitoring methods. The PCR-RFLP methods described in this paper match these criteria and can accurately identify three species of Asian otters. These protocols also have important forensic applications such as the identification of seized otter pelts and roadkills. Accurate species identification is also an important step that should precede genetic identification of individuals, sex, relatedness, population genetics, and diet assessment by metabarcoding. The methods we describe here can be used to generate baseline spatial distribution maps, including studies of occupancy, species interaction and habitat use, genetic monitoring, and other valuable ecological information that will facilitate the conservation of otters in Asia. writing -original draft (equal); writing -review and editing (equal).

ACK N OWLED G M ENTS
We express our gratitude to the following who either contrib-

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.